Preparation and characterization of monoclonal antibodies against GAM protein: a novel gp130-associated molecule

gp130-associated-molecule (GAM) is a recently cloned 24-kDa protein, which binds to gp130 at its cytoplasmic membrane-proximal region and has high homology with the N-terminal of Groucho/TLE molecules, a transcription co-repressor family playing an essential role in Notch signaling. Expression of GAM in COS7 cells inhibited the association of JAKs with gp130, and decreased the tyrosine phosphorylation level of these molecules as well. To further investigate the function of GAM, monoclonal antibodies (MAbs) to GAM were prepared. First, GAM-Thioredoxin(Thio) fusion protein was expressed in E. coli and purified with anti-Thio PAb coupled Sepharose-4B. Using purified GAM-Thio as immunogen, three MAbs against GAM with high affinity were raised by conventional B-lymphocyte hybridoma technique. They could recognize different epitopes of nature and denatured GAM-Thio without any cross-reaction with Thio or components of E. coli or with TLE1-GST fusion protein. In Western blotting and flow cytometric assay, these MAbs can detect cellular GAM protein and verify the increase of GAM expressing in GAM cDNA permanently transfected M1 cells. Furthermore, Western blotting with these MAbs indicated that GAM formed 110 kDa polymers in the nucleus. These MAbs represent powerful in investigating the role of GAM in gp130 signaling and Notch signaling.